Previously known proteins were purified and found to have effects on lipogenesis. Control of these proteins by inhibitors or by antisense molecules, may be used to control obesity and other lipid-production related conditions in mammals.
Obesity is a major health problem in humans because it causes an increased risk for various diseases, including high blood pressure, diabetes, coronary heart disease, stroke, respiratory complications, some forms of cancer, and joint and back problems (Kopelman, 2000). An accepted international definition of an obese individual, is a person whose body mass index (weight divided by the square of the body""s height) is 30 kg per meter square or higher (WHO, 1998). According to this definition, more than 20% of the U.S. population is currently obese. This high percentage is, most likely, due to an increased food intake, encouraged by the abundance, constant advertisement and low-price of high caloric foods.
Although increased food intake is the major contributor of obesity, its level is influenced by the genetic make-up, environment and life style of the affected individual (Hill and Peters, 1998). The influence of heredity can be deduced from the observation that obesity tends to run in families. Yet, better illustrations of the impact of genetics comes from studies of identical twins. Independent of their food intake and environment, such twins exhibit a similar weight gain and similar fat deposition sites (Bouchard et. al., 1990; Allison et. al., 1996). By means of a genome-wide linkage scan, it was recognized that humans have more than 40 potential obesity associated genetic loci (Perusse et. al., 1999), whereas, based on crosses between inbred strains, mice have more than 70 such loci (Barch et al., 2000). Because of the polygenic nature of obesity, the influence of the environment and the differences in life style, it was difficult to directly ascertain in humans the identity of specific obesity genes. Fortunately, because of the availability of obese mouse models and the evolutionary conservation of many genes between mice and humans, it was possible to characterize a number of obesity genes. The most studied of these are the ob gene, which encodes the adipocyte-derived hormone leptin, and the db gene, which encodes its receptor. It appears that the interactions of leptin and insulin with their corresponding receptors in the brain contribute to the regulation of food intake (Schwartz et al., 2000). However, obesity resulting from alterations in the structure of ob or db genes are rare (Montague, et al., 1997; Clement et al., 1997; Strobel et al., 1998). Other obesity genes derived from mouse models are the fat gene, which encodes carboxypeptidase E and the agouti gene, which encodes an antagonist of the alpha melanin-stimulating hormone and is associated with mouse far pigmentation (Barsh et al., 2000).
An important aspect of understanding obesity is identifying the processes that lead to the differentiation of progenitor cells into adipocytes (fat cells) and the characterization of the cellular signaling that brings about such differentiation. The development of immortal-mouse cell culture models during the seventies (Green and Kehinde, 1974) was a major catalyst for such an understanding. Most of the studies with these and related cell culture models focused on the nature and function of transcription factors involved in adipogenesis. Particular emphasis was on the CCAAT/enhancer binding protein family, peroxisome proliferator-activated response elements and adipocyte determination- and differentiation-dependent factors (Brun et al., 1996; Mandrup and Lane, 1997). In addition, these cell culture systems helped identify a number of natural inducers of adipocyte differentiation, predominantly including the insulin-like growth factor xe2x88x921, the growth hormone and a number of corticosteroids (Cornelius et al., 1994; Ailhaud et al., 1994a; Ailhaud et al., 1994b; Smas and Sul, 1994). While these studies have helped to advance obesity research, there is still a need to identify circulatory factors that may directly affect adipocyte differentiation and consequent obesity, the fat reserves in the human body. Once identified, it may be possible by an appropriate inhibitions or regulation of these factors to alleviate obesity and its negative impact on health.
There are several molecules whose existence was previously known, but were not suspected of having any role in adipocyte differentiation. These include the human extracellular matrix protein 1 (ECM-1), human glia-derived nexin I alpha protein (NP-I), human histone H2A (H2A) and human tissue inhibitor of metaloproteinase-2 (TIMP-2).
ECM-1 was originally identified as an 85-kD protein secreted by an osteogenic (MN7) cell line from the mouse (Mathieu et al., 1994). This protein is encoded by a 1.9 kb mRNA, whereas the human form of this protein is encoded by a 1.8 kb mRNA. ECM-1 gene expression was detected in a number of tissues including brain, heart, kidney, lung and muscle. Besides, both mice and humans also have a shorter splice form of the gene; in the mouse a 1.4 kb mRNA and in the human a 1.5 kb mRNA. The shorter human splice form is primarily detected in human tonsils and skin (Bhalerao et al., 1995; Smits et al., 1997). The cellular function of these two splice forms is not known. Yet, recently it was reported that the level of the shorter transcript increases during human keratinocyte differentiation (Smits et al., 2000). An ECM-1 associated polypeptide was suggested for the treatment and/or prevention of skin diseases (U.S. Pat. No. 5,981,220).
Serine endopeptidases use serine as the nucleophile in peptide bond cleavage, and are involved in many cellular functions. (Barerett, 1986). In addition, there are specific protein families that effectively inhibit these proteases. NP-I, which is a glycoprotein with a mass of 43-50 kDa, belongs to such a family of inhibitors, termed serpins (Scott et al., 1983; Mbebi et al., 1999). This inhibitor, which is produced and released by different cells including fibroblasts, myotubes, glial cells and vascular smooth muscle cells, is found in serum but in minute amounts. Biologically, NP-I has been found to induce neuronal differentiation in culture (Diaz-Nido et al., 1992).
The matrix metalloproteinases are a family of zinc-dependent endoproteinases that degrade components of the extracellular matrix. These proteinases and their inhibitors, including TIMP-2, play a key role in normal physiological processes such as developmental programs of different tissues, cell migration, wound healing and angiogenesis as well as in pathological processes such as arthritis, atherosclerosis and tumor cell invasion and metastasis (Bode et al., 2000). As described in the present invention, TIMP-2, has a mass of about 25-kDa (Goldberg et al., 1989).
Histone H2A, which is a small basic protein, is an important component of chromatin composition and structure. In addition, H2A, which has many variants was reported to display hormone-like activities. For example, in anterior pituitary cells it behaves as a gonadotrophin-releasing hormone (Brown et al., 1998), in the thymus it acts as a homeostatic hormone (Reichhard et al., 1985), in luteinized ovarian cells it exhibits an anti-gonadotropic effect (Margolin et al., 1992), whereas in mammary cells it functions as a growth factor (Watanabe et al., 1996). H2A was also reported to induce cellular differentiation in myeloid leukemia cells (Okabe-Kado et al., 1981). In addition, H2A has been recommended for use as a stimulator of the immune response (U.S. Pat. No. 4,818,763).
The present invention describes the surprising identification of ECM-1, NP-1, H2A and TIMP-2 as lipogenins.
The present invention relates generally to the use of human lipogenins including extra-cellular matrix protein 1 (ECM-1), human glia-derived nexin I alpha protein (NP-1) human tissue inhibitor of metalloproteinase-2 (TIMP-2) and human histone H2A (H2A) singly or in various combinations to control lipogenesis. For the present invention a lipogenin is defined as a protein/peptide, or a mixture of proteins and/or peptides that induces cellular lipid (fat) droplet formation, which is due to the production and/or accumulation of cellular lipids in the droplets.
In particular, this invention relates to the use in lipogenesis of purified and isolated DNA sequences that encode ECM-1, NP-I, TIMP-2, or H2A, of expression products of these DNA sequences in transformed or transfected host cells, of recombinant and synthetic proteins and peptides having amino acids deduced from these DNA sequences, and of antibodies specific for such proteins and peptides, in controlling lipogenesis. In addition, this invention relates to the use of ECM-1, NP-I, TIMP-2 or H2A or a portion thereof, for the identification, isolation and characterization of binding sites responsible for the lipogenic effect of ECM-1, NP-I, TIMP-2 or H2A. This invention also relates to the use of lipogenic ECM-1, NP-I, TIMP-2, or H2A proteins or peptides, and the corresponding binding proteins or peptides in assays to identify drugs that modify the lipogenic action of these proteins or peptides. Modification of lipogenesis is a method to control obesity.
By xe2x80x9ccontrolxe2x80x9d is meant herein, affecting the production of lipid droplets over controlled values in cells not control with a lipogenin.